The aims of our studies are (1) to elucidate the role of gene expression of heme pathway enzymes in erythroid cell differentiation, and (ll) to define the molecular nature of enzymatic defects of the heme biosynthetic pathway in patients with inherited forms of porphyrias. The first part can be considered as molecular biological studies in cellular systems, while the second part can be considered as clinical investigations of the molecular defects of heme pathway enzymes in human patients. (I) Our studies have demonstrated (i) differential regulation of genes encoding the erythroid-specific delta-aminolevulinate synthase (ALAS-E), and the non-specific delta-aminolevulinate synthase (ALAS-N) during erythroid differentiation of murine erythroleukemia (MEL) cells, and (ii) sequential gene activation of heme pathway enzymes during erythroid differentiation of these cells. In this renewal proposal, we plan to examine the role of gene expression of erythroid-specific heme pathway isozymes in heme synthesis of normal human erythroid progenitor cells. Recent findings in animal cells suggest that there are several tissue-specific heme pathway isozymes and they may be in a tissue-specific manner. Therefore, we will examine changes in the level of mRNAs encoding the erythroid-specific and the non-specific ALAS, ALA dehydratase (ALAD) and porphobilinogen deaminase (PBGD) in normal human erythroid progenitor cells during erythroid differentiation, to elucidate whether there is distinctive regulatory features of there isozymes in human cells. (II) We have defined the first molecular defects of ALAD and ferrochelatase (FeC) in patients with ALAD deficiency porphyria (ADP) and erythropoietic protoporphyria (EPP), respectively. We will expand our effort to define the molecular nature of enzymatic defects in other porphyrias. We will examine (i) the molecular nature of a unique defect of uropoietic porphyria (HEP) with an abnormally decreased cross-reactive material (CRIM), (ii) the nature of the unique erythroid-specific defect of ALAD in a pedigree with homozygous ADP, (iii) the molecular defects of ferrochelatase in patients with EPP in Japan, and (iv) the gene defects of ALAS in patients with sideroblastic anemia. These studies will undoubtedly shed much light on better understanding of the role of gene expression of the erythroid-specific heme pathway enzymes during erythroid cell differentiation and the consequences of the genetic defects of these enzymes in erythroid heme synthesis. In order to accomplish these goals, necessary collaborations have also been arranged.